The present application relates to an optical measurement apparatus for optically detecting a sample by a flow channel. To put it in detail, the present application relates to an optical measurement apparatus for optically detecting a sample flowing through a flow channel, a flow site meter making use of the optical measurement apparatus and an optical measurement method adopted by the optical measurement apparatus.
In recent years, with progress of an analysis technique, there is also developed a technique for measuring microparticles, analyzing the measured microparticles and sorting the analyzed microparticles in a process of driving the microparticles to flow through a flow channel. In the process for which the technique is developed, biological microparticles or general microparticles are driven to flow through a flow channel. Typical examples of the biological microparticle are a cell and a microorganism whereas a typical example of the general microparticle is a microbead. A typical example of the technique for measuring microparticles flowing through a flow channel, analyzing the measured microparticles and sorting the analyzed microparticles is an analysis technique referred to as a flow-site-metry technique. The technological improvement of flow-site-metry technique is making progress very fast.
The flow-site-metry technique is an analysis technique in accordance with which microparticles serving as the subject of an analysis are driven into fluid in an aligned state, laser light or the like is radiated to the microparticles in order to detect fluorescent or scattered light emitted by the microparticles due to the radiation and, on the basis of the detected fluorescent or scattered light, the microparticles are measured, analyzed and sorted. A flow-site-metry process is carried out in a total system which can be divided into a fluid system, an optical system, an electrical system and a sorting system. The fluid system, the optical system, the electrical system and the sorting system are described as follows.
(1): Fluid System
In the fluid system, the microparticles serving as the subject of an analysis are aligned on a line inside a flow cell (or a flow channel). To put it more concretely, a sheath flow is driven to enter a flow cell at a fixed flow rate and, in this state, a sample flow including the microparticles is injected slowly into the center of the flow cell. At that time, in accordance with the laminar flow principle, the sheath flow and the sample flow do not mix with each other. Instead, the sheath flow and the sample flow form a laminated flow which is a flow including flow layers. Then, in accordance with, among other quantities, the size of each of the microparticles serving as the subject of an analysis, the flow rates of the sheath flow and the sample flow are adjusted in order to drive each of the microparticles in an aligned state.
(2): Optical System
In the optical system, laser light or the like is radiated to the microparticles serving as the subject of an analysis and fluorescent or scattered light emitted by the microparticles due to the radiation is detected. Each of the microparticles put in an aligned state in the fluid system (1) is driven to flow through a laser radiation section and, every time one of the microparticles passes through the laser radiation section, fluorescent or scattered light emitted by the microparticle due to the radiation of the laser light to the microparticle is detected by an optical detector for every parameter in order to analyze the microparticle.
(3): Electrical System
In the electrical system, optical information detected by the optical system (2) is converted into an analog electrical signal which is a train of voltage pulses. The resulting analog electrical signal is then subjected to an analog-to-digital conversion process. Subsequently, on the basis of digital data obtained as a result of the analog-to-digital conversion process, computer software provided for analyses is used to produce an extracted histogram in an analysis.
(4): Sorting System
In the sorting system, the microparticles completing the measurement processes carried out in the fluid system (1), the optical system (2) and the electrical system (3) are separated from each other before being collected. In accordance with a representative sorting method, plus and minus electrical charges are added to the microparticles completing the measurement processes and the flow cell is sandwiched by two deflection plates D which have electrical potentials different from each other. Each of the electrically charged microparticles is attracted by one of the two deflection plates D in accordance with the polarity of the electrical charge added to the microparticle.
A technology such as the flow-site-metry technology for analyzing and sorting microparticles flowing through a flow channel is widely used in a variety of fields such as the medical field, the pharmaceutical field, the clinical examination field, the food field, the agricultural field, the engineering field, the forensic pathological field and the criminal identification field. Particularly, in the case of the medical field, the flow-site-metry technology plays an important role in sub-fields such as pathology, tumor immunology, transplantations, genetics, regenerative medicine and chemotherapy.
As described above, the technology for measuring microparticles flowing through a flow channel, for analyzing the measured microparticles and for sorting the analyzed microparticles is required a very wide variety of fields. A flow-site-metry process adopting the flow-site-metry technique based on this technology is carried out in a total system which can be divided into the fluid system, the optical system, the electrical system and the sorting system as described above. The development of the technology required for carrying out processes in the fluid system, the optical system, the electrical system and the sorting system is making progress from day to day. For example, as disclosed in Japanese Patent Laid-Open No. 2007-101314 (hereinafter referred to as Patent Document 1), there has been proposed a microparticle analysis apparatus in which microparticle suspension liquid including dyed microparticles serving as the subject of an analysis is generated and light is radiated to a flow of the microparticle suspension liquid in order to obtain a plurality of optical signals having different types each reflecting the characteristic of a microparticle. Each of the optical signals is then converted into an output electrical signal so that analyses having a variety of types different from each other can be carried out by the microparticle analysis apparatus even without preparing signal processing circuit boards with a plurality of different types.
As disclosed in JP-T-2007-518991, on the other hand, there has been proposed a method for widening the dynamic range of a flow site meter. In accordance with this method, fluorescent light radiated by a single light source is split into a plurality of fluorescent light beams and, by detecting these fluorescent light beams, the dynamic range of a flow site meter can be increased.
JP-T-2009-063305 discloses a method for eliminating radiation irregularities and shifts of the radiation position as well as shifts of the focal position. In accordance with this method, directional light is radiated to a sample in order to obtain information on the position of the sample in a flow channel. Then, on the basis of the information, directional light is radiated again to the sample in order to eliminate radiation irregularities and shifts of the radiation position as well as shifts of the focal position.